Minimizing off-target frequency of CRISPR/Cas9 systems via zwitterionic polymer conjugation/peptide fusion PROJECT SUMMARY One of the major obstacles that impede the therapeutic and clinical application of CRISPR/Cas9 systems is the high frequency of off-target activity. A single guide RNA (sgRNA) which recognizes 20-bp target DNA sequences can tolerate mismatches at up to 5 nucleotide positions. This suggests that there are up to thousands of off-target sites for a given nuclease in the human genome. Off-target DNA cleavages can lead to mutations at unintended genomic loci and to chromosomal rearrangements. A deeper analysis shows that most off-target events result from the nonspecific mismatch between sgRNA and target DNA. Therefore, minimizing nonspecific RNA-DNA interactions can be an effective solution to this issue. Here, we propose a zwitterionic poly(carboxybetaine) (pCB) polymer or poly(EK) peptide based protein-polymer/peptide conjugation technology to address this mismatch issue. Zwitterionic polymers and peptides are super-hydrophilic and uniquely resistant to nonspecific interactions in complex media. Due to their excellent nonfouling properties, our previous work demonstrated that zwitterionic materials have no negative effect on the activity of nearby or conjugated proteins. Thus, it is hypothesized that the conjugation of CRISPR/Cas9 system with pCB polymer can both increase the bioactivity of Cas9 nuclease and reduce non-specific interactions between gRNA and DNA. Our preliminary work demonstrated that the conjugation of pCB polymers to CRISPR/Cas9 system can greatly reduce the off-target DNA break. By easily adopted chemical conjugation or genetic fusion, the off-target efficiency can be largely decreased without sacrificing the on-target rate. The objective of this proposed work is to study the role of zwitterionic polymers or peptides in reducing the off-target effect of conjugated or fused CRISPR/Cas9 systems. The off-target effect of editing HBB and CCR5 genes will be analyzed due to their significant clinical importance and reported substantial off-target activity. Specific Aims are: 1) Explore the influence of chemically conjugated zwitterionic polymers on the on/off target effect of CRISPR/Cas9 systems for gene editing of HBB and CCR5; and 2) Explore the influence of genetically fused zwitterionic peptide on the on/off target effect of CRISPR/Cas9 systems. Successful completion of this proposal will culminate in a robust and easily used strategy that eliminates off-target mutations on a genome-wide scale, and improve a wide array of CRISPR/Cas9 biotechnological and therapeutic applications.